The purpose of this research is to investigate the molecular mechanisms of action of biologically active proteins from arthropod disease vectors and pathogenic microorganisms. We use biological and physical techniques to characterize and understand the modes of action of pharmacologically active components from the saliva of blood-feeding vector insects and ticks, as well as immunomodulatory components secreted by parasitic organisms such as Toxoplasma and Schistosoma. Proteins and small molecules found in the saliva of vectors inhibit the host hemostatic responses and are essential for the successful completion of a blood meal. Most vector borne diseases are transmitted during feeding, so elucidation of the physiology and biochemistry of this process is necessary for understanding disease transmission. Saliva has also been shown to have pronounced effects on host inflammatory and immune responses which persist after feeding and can dramatically alter the environment for the pathogen after transmission. Determining the specific role of salivary molecules in these processes is essential for the understanding their importance to pathogen survival after transmission Over the past several years we have identified the functions of numerous salivary molecules involved primarily in overcoming host hemostatic defenses. The raw material for these studies comes from the analyses of salivary transcriptomes produced in collaboration with Dr. Jose Ribeiro. Bioinformatic analysis of sequence data is used to predict function of salivary proteins. Candidate proteins are then expressed in bacterial or eukaryotic cell systems. The proteins are purified and assayed using a variety of methods. Functionally characterized proteins are then produced in larger quantity for structural and other biophysical studies. During the 2019 fiscal year we have 1) Continued our initial study of the role of Aedes aegypti hemolymph juvenile hormone binding protein in regulating hemocyte development and antibacterial immunity. 2) Continued the structural analysis of LJL143, a protein inhibitor of the alternative pathway of complement in the sand fly. We have been working on solution of the crystal structure of the inhibitor and the cryo EM structure of the complex of LJL143 with the C3bB proconvertase complex. 3) Completed structural and functional analysis of a platelet aggregation inhibitor protein from the saliva of Phlebotomus sand flies. 4) Identified an extremely potent inhibitor of thrombin from saliva of the rat flea. 5) Collaborated with Dr. Long and her PhD student Erin Coonahan in the characterization of DNA aptamers selected to bind antimalarial drugs. 6) In collaboration with Dr. Valenzuela and Dr. Tiago Serafim isolated a blood serum constituent that promotes aggregation of Leishmania parasites in the sand fly gut and may act to promote recombination. 1) Over the past decade we have shown that the D7 protein family in mosquito saliva functions by sequestering host-produced mediators of hemostasis and inflammation. Among these are the eicosanoids thromboxane A2 and leukotriene C4. Since these mediators are not known to function in the mosquito, the D7 proteins must be derived from ancestors with different function. I have identified mJHBP, a protein that shows sequence conservation between various genera of mosquitoes and is similar to salivary D7s but has changes in amino acid residues important for binding of vertebrate eicosanoid ligands. Analysis of various tissues and life stages showed that this protein is expressed in the fat body and directed to the blood of the insect. Analysis of mJHBP ligand binding using calorimetry showed that the protein binds the important insect hormone, juvenile hormone, with high selectivity. This hormone is essential for metamorphosis, egg development and male mating behavior. In collaboration with Zach Adelman and Eric Calvo we found that the protein is essential for haemocyte development and normal anti-microbial immunity. Adult females of an mJHBP CRISPR knockout show deficiencies in haemocyte proliferation and phagocytosis. This leads to an inability to control bacterial infections and a delay in the expression of antimicrobial peptides. Injection of wild-type protein reverses this phenotype and results normal ability to control bacterial infection. We have further shown that ligand binding is essential for activity of the protein by designing a panel of site-directed mutants in which the binding of juvenile hormone is impaired or completely eliminated. Injection of these mutant proteins into knockout mosquitoes showed that only proteins with hormone binding capability were able to restore normal hemocyte development and immune responses to the deficient line. 2) Inhibition of the complement cascade is an important feature of saliva from blood feeding vectors. Activation of the complement system in host blood can result in the destruction of insect tissues and production of proinflammatory anaphylatoxins. In collaboration with Dr. Valenzuela, we have identified lufaxin, an inhibitor of the alternative pathway of complement in the saliva of the sand fly Lutzomyia longipalpis. To continue this we are working to determine the X-ray crystal structure of LJL143, which will then be used to determine the cryo EM structure of the inhibited C3bB complex. Purification of the protein from HEK 293 cell supernatants yielded over 10 mg /L of protein which produced diffracting crystals. In collaboration with Dr. Natalia De Val at NCI Frederick I have been working to determine the cryo-EM structure of the inhibited C3bB complex. We have determined the structure of the C3bB complex and are working to collect usable data from preparations including the inhibitor. 3) In order to feed normally, blood-feeding insects need to inhibit the hemostatic system of the host. Platelet activation is an early hemostatic response to wounding that acts to stem the loss of blood. Wounding by vector mouthparts leads to platelet activation through contact with exposed collagen, leading to release of secondary agonists which amplify the activation response. We found that D7 proteins from Phlebotomus sand flies bind the secondary agonist thromboxane A2 causing inhibition of activation. We also determined the crystal structure of this protein and explored its evolutionary relationships with related proteins in mosquito saliva. This work was published in Scientific Reports. 4)In addition to the inhibition of platelet activation, vector insects must inhibit the coagulation cascade in order to feed normally. From the transcriptome of the rat flea salivary gland, we have identified a potent inhibitor of thrombin that inhibits the enzyme with a Ki value of less than 100 picomolar. Additionally, the inhibitor is a relatively small peptide of only 36 amino acids that may have pharmaceutical potential in addition to being the first salivary anticoagulant identified from the Siphonaptera, the taxonomic order of fleas. 5) I have collaborated with Dr. Long and Erin Coonahan to use surface plasmon resonance to analyze the binding affinity of the antimalarial drugs piperaquine and mefloquine for DNA aptamers selected by Erin with the goal of developing a practical detection system for these in the blood. 6)Finally, I have collaborated with Dr. Valenzuela and Dr. Serafim to isolate a component of mammalian plasma that promotes aggregation of Leishmania in the sand fly gut and appears to be of key importance in the poorly understood process of genetic recombination in this important parasite.